First Report of Fusarium proliferatum Causing Basal Rot and Dry Rot on Onion Bulbs (Allium cepa) in Spain.

Spain is the second largest onion-producing country in Europe with 1,465,430 tons and an export value of 584 million € in 2021 (MAPA 2022). In summer 2022 rot bulb symptoms were observed in five commercial fields and during the storage of cultivars 'Orlenda', 'Veleta', 'Mallory', 'Citation' and 'Pantano' from La Roda in Albacete (Castilla-La Mancha, Spain). Approximately 20% of sampled bulbs (113 bulbs analyzed) were affected with dry scales showing brown to dark brown rot on the top and basal plate of the onion bulbs. Occasionally, white to light pink fungal mycelium was observed between rotten scales and the plate basal. Sections of dry scales (5-10 mm) of the apical and basal plate were cut and placed on potato dextrose agar (PDA) and Komada medium (Komada 1975). From 5-day-old cultures typical white to light pink mycelium with microconidia in chains formed on polyphialides and macroconidia resembling Fusarium proliferatum (Nelson et al. 1983). To confirm the pathogen identity, partial translation elongation factor 1-alpha (TEF1) and RNA polymerase II subunit 2 (RPB2) genes were amplified and sequenced using primers reported in O´Donnell et al. (1998) and Samuels et al. (2002) for TEF1 and Liu et al. (1999) for RPB2. In BLAST analyses, the sequences showed 100% identity to the corresponding region of F. proliferatum (KP964908 and JF740801). Sequences were submitted to GenBank, and registered accession numbers are OR061014-16 for TEF1 and OR061017-19 for RPB2. Pathogenicity tests were conducted by inoculating healthy onion bulbs (five replicates per treatment) on the apical and basal plate by placing a 7-day old mycelial plug (10 mm diameter) from PDA cultures. Two onion cultivars ('Pandero' by Nunhems USA and 'Mallory' by Bejo The Netherlands) were inoculated separately with three isolates (PRO1, PRO9, PRO12). Control bulbs were inoculated with sterile PDA. The experiment was carried out twice. All bulbs were placed in a moist chamber and incubated at 25°C in the dark. After 15 days, bulbs inoculated with mycelial plugs showed similar symptoms to those of the original diseased bulbs. Browning dry rot was observed on the apical and basal plate of bulbs. When bulbs were cut longitudinally inner progressing rot was observed. Control bulbs remained symptomless. In both experiments, F. proliferatum was successfully re-isolated and morphologically confirmed from symptomatic bulbs to fulfill Koch's postulates. These results confirmed that isolates PRO1, PRO9 and PRO12 were the pathogen causing basal and dry rot on onion bulbs. This pathogen has recently been identified in China on Allium cepa L. var. agrogatum (Liu et al. 2022) and Idaho on onion (Beck et al. 2020) and could become a serious threat to onion production in Spain, reducing the quality and yield of onion.

Evaluation of Trichoderma spp. on Fusarium oxysporum f. sp. asparagi and Fusarium wilt Control in Asparagus Crop.

Among the key diseases affecting the asparagus crop (Asparagus officinalis L.), vascular wilting of asparagus caused by Fusarium oxysporum f. sp. asparagi stands out worldwide. This disease significantly shortens the longevity of the crop and limits economic production. Traditional control measures have been largely ineffective, and chemical control methods are difficult to apply, making biological control approaches, specifically the use of Trichoderma, an economical, effective, and risk-free alternative. This study aimed to identify the main factors that affect the efficacy of biopesticides studied as Biological Control Agents (BCAs) against Fusarium wilt in asparagus and to assess the efficacy of Trichoderma-based biopesticides under greenhouse and semi-field conditions. We evaluated the response of three Trichoderma spp. (T. atroviride, T. asperellum, and T. saturnisporum) to environmental variables, such as temperature and water activity, and their antagonistic capacity against Fusarium oxysporum f. sp. asparagi. All three Trichoderma species inhibited the growth of the pathogen in vitro. A decrease in water activity led to a greater reduction in the growth rate. The efficacy of the three biological control agents decreased with higher temperatures, resulting in minimal inhibition, particularly under conditions of restricted available water in the environment. The effect of the fungal inoculum density was also analyzed at two different temperatures. A direct correlation between the amount of inoculum and the score on the Disease Severity Index (DSI) was observed. A notable reduction in DSI was evident in treatments with high inoculum density (106 conidium/mL) for all three species of Trichoderma tested at both temperatures. In greenhouse and semi-field tests, we observed less disease control than expected, although T. asperellum and T. atroviride showed lower disease severity indices and increased the dry weight of seedlings and crowns, whereas T. saturnisporum resulted in the highest disease rate and lowest dry weight. This work highlights that the efficacy of Trichoderma as BCAs is influenced by various factors, including the quantity of soil inocula, and environmental conditions. The study findings have strong implications for selecting appropriate Trichoderma species for controlling specific pathogens under specific environmental conditions.

First Report of Fusarium oxysporum f. sp. lactucae Race 4 Causing Lettuce Wilt in Spain.

Spain is the fourth largest lettuce-producing country in the world and the leading European producer. Much of the production, mainly grow in open field, is dedicated to export with a value of 887 million U.S. dollars per year. In summer 2021 wilting symptoms were observed in a commercial field crop on butterhead lettuce 'Amible' in Albacete (Castilla-La Mancha, Spain). Approximately 15% of plants were affected, but losses were even more severe on subsequent crops. Vascular tissue of affected plants showed a brown to red discoloration. Sections of infected vascular tissue (3 to 5 mm long) were surface sterilized in 70% ethanol for 30 s, washed three times with sterile water, and plated on potato dextrose agar (PDA) amended with streptomycin sulfate (100 mg/liter). From 5-day-old cultures typical pale cream to purplish mycelia with microconidia, macroconidia, and chlamydospores of Fusarium oxysporum were observed. Microconidia were abundant on carnation leaf agar and measured 6.1 to 9.2 µm (mean 7.1 ± 0.7 µm; n=50)). Macroconidia were sparse, three-septate, straight to slightly curved, 23.3 to 34.8 × 4 to 5.2 µm (mean 31.5 ± 2.8 × 4.2 ± 0.3 µm; n=50). Chlamydospores were terminal and intercalary, rough walled, and measured 7.2 to 10.1 µm (mean 9.5 ± 0.6 µm; n=50) µm. DNA was extracted from three single-spore isolates using the protocol of Querol et al. (1992) and the translation elongation factor 1-α gene (TEF) was sequenced with exTEF-F/FUexTEF-R primers as described by Taylor et al. (2016). All TEF sequences (GenBank accession no. OP903519) were identical. In BLAST analyses, the isolates showed 100% identity to the corresponding region of Fusarium oxysporum f. sp. lactucae (FOL) race 4 (MK059958). All Spanish isolates were identified as FOL race 4 using a race-specific polymerase chain reaction (PCR) with the primers FPUF/FPUR (Gilardi et al. 2017), and a previously identified FOL race 4 isolate Fus 1.01 as a positive control. Pathogenicity tests were conducted to confirm the positive result of the race 4-specific PCR and to complete Koch's postulates. Three differential lettuce cultivars ('Costa Rica No. 4', 'Banchu Red Fire', and 'Romana Romabella 30 CN') provided by Rijk Zwaan (The Netherlands) were inoculated with three Spanish isolates (Al1A1, Al1D, Al2B) and the Fus 1.01 isolate used as FOL race 4 positive control (Claerbout et al., 2018). Roots of 3-week-old plants (five replicates per treatment) were dipped in a spore suspension (1 × 106 conidia/ml) for 10 min before transplanting into 250-ml pots with sterile substrate. Non-inoculated control plants were dipped in sterile water for 10 min. The experiment was carried out twice. Inoculated lettuce seedlings were planted and maintained in a growth chamber (25°C day, 18°C night). Plants were slightly watered every other day. After 21 days, wilting was observed in the cultivars 'Costa Rica No. 4' and 'Romana Romabella 30 CN'. Moreover, taproots were cut longitudinally, and vascular browning was observed in the taproot. No discoloration could be observed in the taproot of 'Banchu Red Fire' plants, coinciding with the result of isolate Fus 1.01 used as FOL race 4 positive control. Non-inoculated control plants remained healthy and vascular browning was not observed. In both experiments, F. oxysporum f.sp. lactucae was consistently reisolated using PDA medium. These results confirmed that the isolates Al1A1, Al1D and Al2B were FOL race 4. This race has recently been identified in The Netherlands (Gilardi et al. 2017), Belgium (Claerbout et al. 2018), United Kingdom, Ireland (Taylor et al. 2019) and Italy (Gilardi et al. 2019) and could become a serious threat to Spain lettuce production.

Fungal Pathogens and Seed Storage in the Dry State.

Seeds can harbor a wide range of microorganisms, especially fungi, which can cause different sanitary problems. Seed quality and seed longevity may be drastically reduced by fungi that invade seeds before or after harvest. Seed movement can be a pathway for the spread of diseases into new areas. Some seed-associated fungi can also produce mycotoxins that may cause serious negative effects on humans, animals and the seeds themselves. Seed storage is the most efficient and widely used method for conserving plant genetic resources. The seed storage conditions used in gene banks, low temperature and low seed moisture content, increase seed longevity and are usually favorable for the survival of seed-borne mycoflora. Early detection and identification of seed fungi are essential activities to conserve high-quality seeds and to prevent pathogen dissemination. This article provides an overview of the characteristics and detection methods of seed-borne fungi, with a special focus on their potential effects on gene bank seed conservation. The review includes the following aspects: types of seed-borne fungi, paths of infection and transmission, seed health methods, fungi longevity, risk of pathogen dissemination, the effect of fungi on seed longevity and procedures to reduce the harmful effects of fungi in gene banks.

Geographical Origin Does Not Modulate Pathogenicity or Response to Climatic Variables of Fusarium oxysporum Associated with Vascular Wilt on Asparagus.

Asparagus crop is distributed worldwide, covering very different climatic regions. Among the different diseases that affect asparagus, vascular Fusarium wilt, caused by Fusarium oxysporum f. sp. aparagi (Foa), stands out. It is not only the cause of large economic losses due to a decrease in yield and shortened longevity of the plantation, but also prevents replanting. This work aimed to determine if F. oxysporum isolates associated with vascular wilt on asparagus have adapted differentially to the different agro-environmental conditions. The potential correlation between origin and mycelial growth under different temperatures and humidity conditions was analysed for isolates from asparagus fields cultivated in northern and southern Europe. The genetic and pathogenic variability were also analysed. While a clear effect of water activity on mycelial growth was observed, all isolates responded in a similar way to changes in water activity in the medium, regardless of their geographical origin. The results revealed a low genetic variability of F. oxysporum isolates associated with vascular wilt on asparagus without signs of differentiation correlated to geographical origin. The southernmost isolates of the two cultivated varieties inoculated did not express more pathogenicity than those isolated from the colder region.

Incidence and Etiology of Postharvest Fungal Diseases Associated with Bulb Rot in Garlic (Alllium sativum) in Spain.

In recent years, different postharvest alterations have been detected in garlic. In many cases, the symptoms are not well defined, or the etiology is unknown, which further complicates the selection of bulbs during postharvest handling. To characterize the different symptoms of bulb rot caused by fungi, garlic bulb samples were collected from six Spanish provinces in two consecutive years. Eight different fungal species were identified. The most prevalent postharvest disease was Fusarium dry rot (56.1%), which was associated with six Fusarium species. Fusarium proliferatum was detected in more than 85% of symptomatic cloves, followed by F. oxysporum and F. solani. Pathogenicity tests did not show a significant correlation between virulence and mycotoxin production (fumonisins, beauvericin, and moniliformin) or the mycelial growth rate. Penicillium allii was detected in 12.2% of the samples; it was greatly influenced by the harvest season and garlic cultivar, and three different morphotypes were identified. Stemphylium vesicarium and Embellisia allii were pathogenic to wounded cloves. Some of the isolated fungal species produce highly toxic mycotoxins, which may have a negative impact on human health. This work is the first to determine the quantitative importance, pathogenicity, and virulence of the causative agents of postharvest garlic rot in Spain.

Fusarium Consortium Populations Associated with Asparagus Crop in Spain and Their Role on Field Decline Syndrome.

Asparagus Decline Syndrome (ADS) is one of the main phytosanitary problems of asparagus crop worldwide. Diseased plants and soil samples from 41 fields from three main production areas of Spain were surveyed. Eight Fusarium species belonging to seven species complexes were identified in soils: F. oxysporum, F. proliferatum, F. redolens, F. solanisensu stricto, F. equiseti, F. culmorum, F. compactum and F. acuminatum. Fusarium oxysporum was the most prevalent species. Statistical correlation (R2 = 88%) was established between F. oxysporum inoculum density and the average temperature of the warmest month. A relationship was also established between three crop factors (average temperature, crop age and F. oxysporum inoculum density) and field disease indices. Significant differences were observed between the distribution of F. oxysporum propagules in white and green asparagus fields. Thirteen Fusarium species belonging to seven species complexes were identified from roots of diseased plants, being F. oxysporum the most prevalent. F. proliferatum, F. oxysporum and F. redolens showed pathogenicity to asparagus and were the main species associated to ADS. Fusarium oxysporum was the species with the highest genetic diversity displaying 14 sequence-based haplotypes with no geographic differentiation. This work contributes to understanding the Fusarium complex associated to ADS for developing accurate integrated disease management strategies.

Multiplex Fluorescent, Activity-Based Protein Profiling Identifies Active α-Glycosidases and Other Hydrolases in Plants.

With nearly 140 α-glycosidases in 14 different families, plants are well equipped with enzymes that can break the α-glucosidic bonds in a large diversity of molecules. Here, we introduce activity-based protein profiling (ABPP) of α-glycosidases in plants using α-configured cyclophellitol aziridine probes carrying various fluorophores or biotin. In Arabidopsis (Arabidopsis thaliana), these probes label members of the GH31 family of glycosyl hydrolases, including endoplasmic reticulum-resident α-glucosidase-II Radial Swelling3/Priority for Sweet Life5 (RSW3/PSL5) and Golgi-resident α-mannosidase-II Hybrid Glycosylation1 (HGL1), both of which trim N-glycans on glycoproteins. We detected the active state of extracellular α-glycosidases such as α-xylosidase XYL1, which acts on xyloglucans in the cell wall to promote cell expansion, and α-glucosidase AGLU1, which acts in starch hydrolysis and can suppress fungal invasion. Labeling of α-glycosidases generates pH-dependent signals that can be suppressed by α-glycosidase inhibitors in a broad range of plant species. To demonstrate its use on a nonmodel plant species, we applied ABPP on saffron crocus (Crocus sativus), a cash crop for the production of saffron spice. Using a combination of biotinylated glycosidase probes, we identified and quantified 67 active glycosidases in saffron crocus stigma, of which 10 are differentially active. We also uncovered massive changes in hydrolase activities in the corms upon infection with Fusarium oxysporum using multiplex fluorescence labeling in combination with probes for serine hydrolases and cysteine proteases. These experiments demonstrate the ease with which active α-glycosidases and other hydrolases can be analyzed through ABPP in model and nonmodel plants.

Fusarium proliferatum - Causal agent of garlic bulb rot in Spain: Genetic variability and mycotoxin production.

Fusarium proliferatum is a world-wide occurring fungal pathogen affecting several crops included garlic bulbs. In Spain, this is the most frequent pathogenic fungus associated with garlic rot during storage. Moreover, F. proliferatum is an important mycotoxigenic species, producing a broad range of toxins, which may pose a risk for food safety. The aim of this study is to assess the intraspecific variability of the garlic pathogen in Spain implied by analyses of translation elongation factor (tef-1α) and FUM1 gene sequences as well as the differences in growth rates. Phylogenetic characterization has been complemented with the characterization of mating type alleles as well as the species potential as a toxin producer. Phylogenetic trees based on the sequence of the translation elongation factor and FUM1 genes from seventy nine isolates from garlic revealed a considerable intraspecific variability as well as high level of diversity in growth speed. Based on the MAT alleles amplified by PCR, F. proliferatum isolates were separated into different groups on both trees. All isolates collected from garlic in Spain proved to be fumonisin B1, B2, and B3 producers. Quantitative analyses of fumonisins, beauvericin and moniliformin (common secondary metabolites of F. proliferatum) showed no correlation with phylogenetic analysis neither mycelial growth. This pathogen presents a high intraspecific variability within the same geographical region and host, which is necessary to be considered in the management of the disease.

Stemphylium Leaf Blight of Garlic (Allium sativum ) in Spain: Taxonomy and In Vitro Fungicide Response.

The most serious aerial disease of garlic is leaf blight caused by Stemphylium spp. Geographical variation in the causal agent of this disease is indicated. Stemphylium vesicarium has been reported in Spain, whereas S. solani is the most prevalent species recorded in China. In this study, Stemphylium isolates were obtained from symptomatic garlic plants sampled from the main Spanish production areas. Sequence data for the ITS1-5.8S-ITS2 region enabled assignation of the isolates to the Pleospora herbarum complex and clearly distinguished the isolates from S. solani. Conidial morphology of the isolates corresponded to that of S. vesicarium and clearly discriminated them from S. alfalfae and S. herbarum on the basis of the size and septation pattern of mature conidia. Conidial morphology as well as conidial length, width and length:width ratio also allowed the Spanish isolates to be distinguished from S. botryosum and S. herbarum. Control of leaf blight of garlic is not well established. Few studies are available regarding the effectiveness of chemical treatments to reduce Stemphylium spp. incidence on garlic. The effectiveness of nine fungicides of different chemical groups to reduce Stemphylium mycelial growth in vitro was tested. Boscalid + pyraclostrobin (group name, succinate dehydrogenase inhibitors + quinone outside inhibitors), iprodione (dicar-boximide), and prochloraz (demethylation inhibitors) were highly effective at reducing mycelial growth in S. vesicarium with EC50 values less than 5 ppm. In general, the effectiveness of the fungicide was enhanced with increasing dosage.